The government may own rights in this application pursuant to grants from The National Institutes of Health including grant number R37 CA 48023
I. Field of the Invention
The present invention relates generally to the field of cellular and molecular immunology. In particular, the present invention relates to the production of monoclonal antibodies in an avian system, e.g., chickens.
II. Related Art
Monoclonal antibodies are valuable tools in basic research and have growing importance for medical diagnosis and therapy. The procedures for making monoclonal antibodies routinely use immunized mice, rats, hamsters or rabbits as the source of antibody producing B cells. Isolated B cells are fused to a nonsecreting, immortalized cell line, and then selected and screened for specific antibody production. A hybridoma will secrete a single monoclonal antibody indefinitely (Kohler and Milstein, 1974). Hybridoma technologies have important limitations, however. In particular, it has been difficult to make antibodies to some proteins and other antigens; it is hypothesized that these molecules have highly conserved epitopes that are not recognized as "foreign" making them less immunogenic in closely related species (Cinader, 1960).
Distinctive features of chicken immunology can be used to advantage to address some of these problems. Chickens can mount an effective immune response to highly conserved mammalian proteins and produce high affinity antibodies (Horton et al., 1984; Larsson et al., 1993). Polyclonal antibodies to calf thymus RNA polymerase II (Carroll and Stoller, 1983) and to the (x-subunit of the rat insulin receptor (Song et al., 1985) were easily made in the chicken; the same antigens were not effective at inducing specific antibody production in the rabbit. Chicken antibodies to 1,25-dihydroxyvitamin D were more specific and less cross-reactive than rabbit antibodies (Bauwens et al., 1987), making it likely that the chicken is recognizing different epitopes. Chicken antibodies made by individual investigators and harvested from egg yolk have been used to study human insulin (Lee et al., 1991), human plasma kallikein (Burger et al., 1985), IL-6 (Wooley et al., 1995), prostaglandins (Fertel et al., 1981) and PCNA (Gassman et al., 1990). In addition, chicken serum containing polyclonal antibodies to human complement components, human erthropoietin, fibrinogen and fibronectin are commercially available (American Research Products, Inc. 1995 Catalogue). However, there is not yet a reliable way to produce chicken monoclonal antibodies given the tendency of chicken cell lines to cease antibody production after a short period of time (Nishinaka et al., 1989, 1991).
Another approach to generating unique antibody sequences recently has developed. It now is possible to clone DNA encoding a specific antibody's heavy and light chains, and to express the cloned antibody chains following transfection into other eukaryotic cells (Neuberger et al., 1983; Gilles et al., 1983). Like hybridomas, these transfected cells can be selected, screened and cloned as stable transfectomas that secrete a monoclonal antibody (Sharon et al., 1984; Morrison, 1985). In addition, the ability to clone the DNA of individual leader (L), variable (V), diversity (D) and joining (J) gene segments (Larrick et al., 1989; Orlandi et al., 1989; Heinrichs et al., 1995) and to manipulate these domain segments by specific mutation and random combination has facilitated the engineering of "artificial" antibody combining sites to a variety of epitopes that can be expressed in transfectomas or by phage display (McMafferty et al., 1990; Winter and Milstein, 1991; Huston et al., 1988; Yamanaka et al., 1996)). These techniques have proved successful in producing monoclonal antibody-type reagents with new specificities and with modified effector functions. Even where successful immunization with a given antigen can be achieved, however, it is technically challenging to identify, and then clone, the heavy and light chain sequences encoding the specific antibody (see Antibody Engineering Chapters 2, 3, 6 Borrebaeck).
Thus, it is clear that there remains a need for improved methods for the production of monoclonal antibodies against epitopes conserved in mammalian species. In addition, there is a need for improved methods in the cloning and manipulation of immunoglobulin genes in various animal species, and in particular, avian species such as chicken.